Expression of the endocannabinoid system and response to cannabinoid components by the human fetal testis.

BACKGROUND: Cannabis consumption by pregnant women continues to increase worldwide, raising concerns about adverse effects on fetal growth and deleterious impacts on the newborn, in connection with evidence of placental transfer of cannabis compound. Cannabis action is mediated by the endocannabinoid system (ECS), which expression is well established in the brain but unknown in the developing testis. The fetal testis, whose endocrine function orchestrates the masculinization of many distant organs, is particularly sensitive to disruption by xenobiotics. In this context, we aimed to determine whether cannabis exposure has the potential to directly impact the human fetal testis. METHODS: We determined the expression of components of the ECS in the human fetal testis from 6 to 17 developmental weeks and assessed the direct effects of phytocannabinoids Δ9-trans-tetrahydrocannabinol (THC) and cannabidiol (CBD) on the testis morphology and cell functions ex vivo. RESULTS: We demonstrate the presence in the human fetal testis of two key endocannabinoids, 2-arachidonylglycerol (2-AG) and to a lower level anandamide (AEA), as well as a range of enzymes and receptors for the ECS. Ex vivo exposure of first trimester testes to CBD, THC, or CBD/THC [ratio 1:1] at 10-7 to 10-5 M altered testosterone secretion by Leydig cells, AMH secretion by Sertoli cells, and impacted testicular cell proliferation and viability as early as 72 h post-exposure. Transcriptomic analysis on 72 h-exposed fetal testis explants revealed 187 differentially expressed genes (DEGs), including genes involved in steroid synthesis and toxic substance response. Depending on the molecules and testis age, highly deleterious effects of phytocannabinoid exposure were observed on testis tissue after 14 days, including Sertoli and germ cell death. CONCLUSIONS: Our study is the first to evidence the presence of the ECS in the human fetal testis and to highlight the potential adverse effect of cannabis consumption by pregnant women onto the development of the male gonad.

Ibuprofen is deleterious for the development of first trimester human fetal ovary ex vivo.

STUDY QUESTION: Does ibuprofen use during the first trimester of pregnancy interfere with the development of the human fetal ovary? SUMMARY ANSWER: In human fetuses, ibuprofen exposure is deleterious for ovarian germ cells. WHAT IS KNOWN ALREADY: In utero stages of ovarian development define the future reproductive capacity of a woman. In rodents, analgesics can impair the development of the fetal ovary leading to early onset of fertility failure. Ibuprofen, which is available over-the-counter, has been reported as a frequently consumed medication during pregnancy, especially during the first trimester when the ovarian germ cells undergo crucial steps of proliferation and differentiation. STUDY DESIGN, SIZE, DURATION: Organotypic cultures of human ovaries obtained from 7 to 12 developmental week (DW) fetuses were exposed to ibuprofen at 1-100 µM for 2, 4 or 7 days. For each individual, a control culture (vehicle) was included and compared to its treated counterpart. A total of 185 individual samples were included. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian explants were analyzed by flow cytometry, immunohistochemistry and quantitative PCR. Endpoints focused on ovarian cell number, cell death, proliferation and germ cell complement. To analyze the possible range of exposure, ibuprofen was measured in the umbilical cord blood from the women exposed or not to ibuprofen prior to termination of pregnancy. MAIN RESULTS AND THE ROLE OF CHANCE: Human ovarian explants exposed to 10 and 100 µM ibuprofen showed reduced cell number, less proliferating cells, increased apoptosis and a dramatic loss of germ cell number, regardless of the gestational age of the fetus. Significant effects were observed after 7 days of exposure to 10 µM ibuprofen. At this concentration, apoptosis was observed as early as 2 days of treatment, along with a decrease in M2A-positive germ cell number. These deleterious effects of ibuprofen were not fully rescued after 5 days of drug withdrawal. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study was performed in an experimental setting of human ovaries explants exposed to the drug in culture, which may not fully recapitulate the complexity of in vivo exposure and organ development. Inter-individual variability is also to be taken into account. WIDER IMPLICATIONS OF THE FINDINGS: Whereas ibuprofen is currently only contra-indicated after 24 weeks of pregnancy, our results points to a deleterious effect of this drug on first trimester fetal ovaries ex vivo. These findings deserve to be considered in light of the present recommendations about ibuprofen consumption pregnancy, and reveal the urgent need for further investigations on the cellular and molecular mechanisms that underlie the effect of ibuprofen on fetal ovary development.

Parallel assessment of the effects of bisphenol A and several of its analogs on the adult human testis.

STUDY QUESTION: Are bisphenol A (BPA) and BPA analogs (BPA-A) safe for male human reproductive function? SUMMARY ANSWER: The endocrine function of human testes explants [assessed by measuring testosterone and insulin-like factor 3 (INSL3)] was impacted by exposure of the human adult testis explants to BPA/BPA-A. WHAT IS KNOWN ALREADY: The few epidemiologic studies performed suggest that bisphenols have potential endocrine disruptive properties, but they did not identify clear and direct patterns of endocrine disruption. STUDY DESIGN, SIZE, DURATION: Adult human testis explants in culture were exposed to BPA and the analogs bisphenol F (BPF), bisphenol S (BPS), bisphenol E (BPE), bisphenol B (BPB) and bisphenol A diglycidyl ether (BADGE) at 10-9-10-5 M for 24 or 48 h. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human adult testes were obtained from prostate cancer patients who had no hormone therapy, or from multiorgan donors. After ex vivo exposure to the investigated bisphenols, the measured outcomes were related to histopathology (gross morphology and germ cell viability determined by anti-caspase three immunohistochemistry), and the levels of testosterone, INSL3 and inhibin B were measured using immunoassays. The levels of mRNA encoding key enzymes of bisphenol biotransformation were investigated by quantitative PCR: UGT2B15 UDP (glucuronosyltransferase two family, polypeptide B15), GUSB (glucuronidase beta), SULT1A1 and 3 (sulfotransferase family 1 A member 1 and 3) and STS (steroid sulfatase). MAIN RESULTS AND THE ROLE OF CHANCE: A significant dose-dependent inhibition was found between testosterone levels measured in the culture medium and concentrations of BPA (P = 0.00778 at 24 h and P = 0.0291 at 48 h), BPE (P = 0.039) and BPF (P = 0.00663). The observed BPA and BPA-A-induced inhibition of testosterone production varied according to duration of exposure and BPA/BPA-A concentrations. BPA (10-9 M; P < 0.05), BPB (10-9 M; P < 0.05), BPS (10-9 and 10-8 M; P < 0.05) and BADGE (10-5 M; P < 0.05) increased Leydig cell INSL3 production. By contrast, BPE dose dependently inhibited INSL3 (P = 0.0372). Conversely, Sertoli cell function (inhibin B) and germ cell viability were not significantly affected by either bisphenols. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Environmental compounds cannot be deliberately administered to men, justifying the use of an ex vivo approach. A relatively low number of testes samples were available for analysis (n = 3, except for testosterone secretion with n = 5). The active concentrations of BPA and BPA-A used in the study were higher than those found in human biological fluids. WIDER IMPLICATIONS OF THE FINDINGS: Under our experimental conditions, direct exposure to BPA or BPA-A can result in endocrine disturbance in the adult human testis. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Inserm (Institut National de la Santé et de la Recherche Médicale), EHESP-School of Public Health, University of Rennes1, by grants from the Agence Nationale de la Recherche (ANR; grant#ANR-13-CESA-0012-03 NEWPLAST) and Agence Nationale de Sécurité Sanitaire de l'Alimentation, de l'Environnement et du Travail (ANSES; grant#EST-2010/2/046 (BPATESTIS)). All authors declare they have no current or potential competing financial interests.

Claudin 11 deficiency in mice results in loss of the Sertoli cell epithelial phenotype in the testis.

Tissue integrity relies on barriers formed between epithelial cells. In the testis, the barrier is formed at the initiation of puberty by a tight junction complex between adjacent Sertoli cells, thereby defining an adluminal compartment where meiosis and spermiogenesis occur. Claudin 11 is an obligatory protein for tight junction formation and barrier integrity in the testis. It is expressed by Sertoli cells, and spermatogenesis does not proceed beyond meiosis in its absence, resulting in male sterility. Sertoli cell maturation--arrest of proliferation and expression of proteins to support germ cell development--parallels tight junction assembly; however, the pathophysiology underlying the loss of tight junctions in the mature testis remains largely undefined. Here, using immunohistochemistry and microarrays we demonstrate that adult Cldn11(-/-) mouse Sertoli cells can proliferate while maintaining expression of mature markers. Sertoli cells detach from the basement membrane, acquire a fibroblast cell shape, are eliminated through the lumen together with apoptotic germ cells, and are found in epididymis. These changes are associated with tight junction regulation as well as actin-related and cell cycle gene expression. Thus, Cldn11(-/-) Sertoli cells exhibit a unique phenotype whereby loss of tight junction integrity results in loss of the epithelial phenotype.